We performed (in collaboration with NCATS) a genome-wide high-throughput siRNA screen of 21,500 cellular genes to identify ones involved in RSV infection. Human airway epithelial A549 cells were engineered to express Ds-Red as a viability marker, and were infected with RSV expressing enhanced green fluorescent protein (GFP) as a marker for viral gene expression. Three siRNAs per cellular gene were evaluated individually for reduction in GFP expression as a measure of inhibition of RSV infection. Computer analysis identified and corrected siRNA off-target effects. The top 155 hits (i.e., the greatest reductions in GFP without excessive loss of viability) were re-evaluated with at least three additional siRNAs each, and the top 65 hits were identified and confirmed manually. Among the genes identified for further analysis, four encoded proteins associated with integrin signaling: actin-related protein 2, ARP2; ADP-ribosylation factor 1, ARF1; integrin alpha-V, ITGAV; and mitogen-activated protein kinase 3, MAPK3. We initially focused on ARP2 and reported those results last year. Briefly, in studies in cell culture, ARP2 was found to be involved in the production and spread of progeny RSV particles to neighboring cells. One role of ARP2 was exemplified by the finding that its expression was needed for efficient production of progeny RSV particles: when expression of ARP2 was knocked down, there was a decrease in the accumulation of virions on the cell surface and in the release of virions into the medium. A second role of ARP2 involved the new finding made in this study that RSV infection greatly increased the formation of filopodia, which are long slender projections from the cell surface. Progeny virions were found to be associated with the filopodia, and contact between these RSV-carrying filopodia and neighboring cells appeared to deliver virus to these neighbors. In addition, RSV infection resulted in increased cell motility in A549 cells, which also increased the delivery of progeny RSV to neighboring cells. Knockdown of ARP2 expression strongly reduced filopodia formation and motility, indicating a role for ARP2 in both processes. In this study, we used multicolor Stimulated Emission Depletion (STED) microscopy to generate high-resolution images of RSV infected cells and particles, and this was described in a Bio-Protocol report that appeared this year. In other experiments presently in progress, we used siRNAs to knock down expression of the other three integrin pathway candidates noted above, namely ARF1, ITGAV, and MAPK3. Knockdown of each of these three proteins reduced the production of progeny RSV, and the effect was increased if ARP2 was simultaneously knocked down. Two of the proteins, namely ARF1 and ITGAV, appeared reduce the efficiency of RSV entry. Further studies of the underlying mechanism are currently under way. Yet another of the 65 top hits in the siRNA screen is a protein that is part of a transmembrane ion channel: we presently are preparing a manuscript on a study characterizing the role of this complex in RSV infection, and will report these results in next year's report. We prepared a number of candidate live-attenuated RSV vaccine viruses, using reverse genetics. Attenuation was achieved by deletion of accessory protein genes and the use of point mutations that were previously developed and had been designed to be refractory to de-attenuation. In particular, we focused on candidates attenuated by deletion of the M2-2 ORF (delM2-2) or the NS1 or NS2 gene (delNS1, delNS2). Each lineage looked very promising in pre-clinical studies, including evaluation in an in vitro model of human airway epithelium (HAE) and in non-human primates. Lead candidates have been manufactured as clinical trial material for Phase 1 clinical studies described in an accompanying report (Clinical Trials of Vaccines for Respiratory Syncytial Virus and Related Viruses). We evaluated MPV as a vector for expressing protective antigen from a heterologous viral pathogen, using the fusion F protein of RSV as a proof of principle. Like RSV, MPV is a member of the Genus Orthopneumvirus, Family Pneumoviridae. RSV and MPV are very similar with regard to genome organization and the numbers and types of genes and encoded proteins, and the two viruses share 16% to 60% amino acid sequence identity, depending on the protein. We previously reported that humans do not appear to be exposed to MPV, based on analysis of a panel of adult and pediatric sera. We also showed that the replication of MPV is highly restricted in the respiratory tract of African green monkeys and rhesus macaques, indicative of a strong host range restriction in primates. We also found an absence of significant antigenic reactivity between human or non-human primate RSV-specific serum antibodies and MPV, and a lack of cross-protection in mice between RSV and MPV. (RSV is the human virus that is the most closely related to MPV and thus would have been the one most likely to cross-react, but this did not appear to occur to a significant extent.) These properties indicated that MPV should be highly attenuated in humans, and the lack of cross-protection with RSV indicated that previous infection with RSV should not restrict an MPV vector. Since other human viruses are much less related to MPV, they also should not be a factor. We evaluated MPV as an RSV vaccine vector. The RSV F ORF was codon optimized and the encoded RSV F protein was made identical to an early passage of RSV strain A2. The RSV F ORF was placed under the control of MPV transcription signals and inserted at the first (rMPV-F1), third (rMPV-F3), or fourth (rMPV-F4) gene position of a version of the MPV genome that contained a codon-pair optimized L polymerase gene. The recovered viruses replicated in vitro as efficiently as the empty vector, with stable expression of RSV F protein as determined by plaque assay and double-immunostaining with antibodies to the vector and to RSV F. Replication and immunogenicity of rMPV-F1 and rMPV-F3 were evaluated in rhesus macaques following intranasal and intratracheal administration. Both viruses replicated to low levels in the upper and lower respiratory tract, maintained stable RSV F expression, and induced RSV-neutralizing serum antibodies at high levels similar to those induced by a 10-fold higher dose of wild-type RSV. In conclusion, this study demonstrated that MPV provides a highly attenuated yet immunogenic vector for the expression of RSV F protein, with potential application in RSV-nave and RSV-experienced populations. This study was performed with unmodified RSV F protein in order to make a direct comparison with wt RSV, and the immunogenicity of this insert should be greatly increased and improved if the protein is modified to be stabilized in the prefusion conformation and to be packaged into the vector particle, using strategies described for parainfluenza virus vectors in the accompanying report Laboratory and Pre-clinical Studies of Parainfluenza Viruses.